The N-methyl-D-aspartate (NMDAR) receptor is the main glutamate receptor subtype, usually participating in rapid excitatory synaptic transmission. These receptors are broadly expressed and have been implicated in physiological processes such as the neuron development, synaptic plasticity, memory and learning and many pathological conditions (Citri and Malenka, 2008). NMDAR has also been involved processes such as ischemic damage (Hardingham and Bading, 2010), chronic pain, psychosis, and the main degenerative disorders such as Parkinson's and Alzheimer's diseases (Mony et al., 2009a; Traynelis et al., 2010).
NMDA receptors are presented as a set of tetramers from two glycine binding GluN1 subunits and two glutamate binding subunits, of which there are four types (GluN2A, GluN2B, GluN2C and GluN2D). Both types of GluN2A and GluN2B subunits are considered the main elements for NMDA receptor functioning in central nervous system (CNS) neurons (Madden, 2002).
The GluN2 subunit controls a broad range of NMDA receptor functional properties and is differentially expressed throughout the whole CNS (Akazawa et al., 1994; Monyer et al., 1994). Each NMDA receptor subunit has four domains: the extracellular amino-terminal domain (ATD), the extracellular ligand binding domain (LBD), the transmembrane domain (TMD) and the intracellular carboxyl-terminal domain (CTD) (Sobolevsky et al., 2009).
Activating NMDA receptors requires two coincident events: glutamate and glycine binding and the simultaneous membrane depolarisation, thereby eliminating Mg2+ channel pore-blocking, giving rise to Ca2+ inflow. Ca2+ inflow (in physiological conditions) produces partial inhibition of NMDA receptors through calcium-dependent inactivation, thereby impeding intracellular Ca2+ overload (Krupp et al., 1999). However, in pathological conditions such NMDA receptor regulation becomes deactivated, resulting in an excess of Ca2+ inflow via the NMDA receptor, thereby triggering multiple intracellular catabolic processes and thus inducing neuron death (Lipton et al., 2006).
Given the NMDA receptor's complex functioning in synaptic transmission, memory and learning and the fact that it is highly implicated in pathological processes such as ischemic damage, pharmacology related to this receptor's regulation has been focused on testing antagonists directed against the glutamate binding site, the glycine binding site, the cannel in question (Mg2+) and the receptor's allosteric regulatory sites, all as neuroprotective agents in many preclinical assays; however, such approaches have failed (Green, 2002; Parsons et al., 2002; Lo et al., 2003; Hoyte et al., 2004; Small and Tauskela, 2005; Wang and Shuaib, 2005; Muir, 2006). Regarding channel blocking agents (aptiganel, cerestat; CNS 1102) and the glutamate binding site, the levels of antagonism needed for producing/inducing neuroprotection affect cardiovascular function and alter cognition (psychotic effects) (Small and Tauskela, 2005; Muir, 2006). Gavestinel (GV150526), directed against the glycine binding site has also failed to provide the desired neuroprotection (Sacco et al., 2001). A selective antagonist from the G1uN2B CP-101,606 subunit is apparently insufficient for protection against severe ischemic damage (Yurkewicz et al., 2005). Regarding other types of pathology, such as Alzheimer's and Parkinson's diseases, memantine has had promising results given its low affinity and rapid dissociation kinetics (classic antagonists lack such characteristics) (Kotermanski and Johnson, 2009).
Other types of antagonist derived from natural poisons have been tested against the NMDA receptor. Conotoxins are one such type of poisons, being small peptides produced by marine invertebrates from the genus Conus. These conotoxins are rigid protein compounds which are cysteine-rich (4-6 residues) in very well-defined positions and synthesised by a complex mechanism facilitating their great variability and efficiency in paralysing their prey, thereby guaranteeing this specie's evolutionary success (Olivera et al., 1997). The conotoxins have been characterised by having enormous specificity, binding to well-defined receptors on muscle or nerve cells where they act as ion channel antagonists, blocking their functionality.
The conantokins (one type of conotoxin) are small peptides (17-27 amino acids long), poor in disulphide bridges, differing from the other conotoxins; they are found in poison from Conus geographus and have high affinity for blocking the NMDA receptor and a potential anticonvulsant and antinociceptive effect (Layer et al., 2004; Xiao et al., 2008). Conantokin-G (CGX-1007), or Con-G, is found within this group; it is a 17 amino acid-long peptide (GEγγLQγNQγLIRγKSN-NH2 (SEQ ID NO: 1)) which is characterised by having five modified gamma carboxyglutamate acid (γ-carboxyglutamic or Gla) aminoacid residues. This toxin has competitive and non-competitive antagonism against NMDA receptor subunits (Prorok and Castellino, 2007). The γ-carboxyglutamic residues enable coordination of divalent ions (mainly Ca2+), thereby conferring α-helix structure on conantokin-G (Myers et al., 1990).
Various approaches involving conantokin-G analogous peptides, in which γ-carboxyglutamic residues have been partially and/or totally replaced by alanine and especially by glutamate (Lin et al., 1999; Chandler et al., 1993), have led to determining that conantokin-G antagonist activity against NMDA receptors depends strongly on amino-terminal residues where γ-carboxyglutamic residues play a structural and functional role, especially residues Gla3 and Gla4 (Blandl et al., 1998; Zhou et al., 1996; Warder et al., 1998).
Along with γ-carboxyglutamic residues, conantokin-G Leucine 5 (Leu 5) its been described to be the determinant molecular allowing this toxin to have NMDA receptor GluN2B subunit LBD specificity (residue Met 739, located in LBD domain D2), thereby producing high selectivity and competitive antagonism against this subunit (Donevan and McCabe., 2000; Sheng et al., 2010).
The role of conantokin-G as antagonist of NMDA receptors has been evaluated in different scenarios. The rol of Conantokine-G as a NMDA receptor antagonist have been evaluated in different scenarios, boosting a large amount of patents. For instance, patent CA 2288346 A1 describes a method in which using Conantokine-G produces analgesia and neuroprotection when administered to mammals. Patent U.S. Pat. No. 5,830,998A discloses a series of peptides base don modifications in N y C terminals of Conantokine-G and other peptides in which γ-carboxyglutamic residues are replaced with glutamate and the use as allosteric modulators of NMDA receptors in the central nevous System disorders treatment. Patent U.S. Pat. No. 6,110,894A uses the same Conantokine-G derivates in the treatment of excitotoxicity produced by the stimulation of NMDA receptor during epilepsy episodes. Recently, patent request CN102167729A describes a Conantokine-G analogue (Glu-instead of-G) where the γ-carboxyglutamic residues are replaced with glutamate, showing an effect on the psychological and physical dependence on morphine; and compared to morphine the analogue has a stronger analgesic effect. It has been found that conantokin G has neuroprotective effects in an ischemic event and in staurosporin-induced apoptosis (Williams et al., 2002).
However, conantokin G's neuroprotective effect regarding an excitotoxic context, particularly concerning ischemia, has not been well established. A recent study concerning organotypic hippocampus cultures and in HEK293 cells expressing different combinations of NMDA receptor subunits (Alex et al., 2011), has highlighted conantokin G's neuroprotective effect in a excitotoxic environment. Conantokin acted as an effective GluN2B and GluN2A subunit blocker in the aforementioned study, suggesting that conantokin G is a potent molecule having a neuroprotective effect regarding an excitotoxic setting and that such effect is mediated by different NMDA receptor subunits, as opposed to previously described studies concerning conantokin-G selectivity for the GluN2B subunit.
Recent data have shown that Conantokine-G promotes neuronal integrity related neuroprotection and changes in the subunits cell location of the NMDA receptor in a in-vivo ischemia model (Balsara et al., 2015)
However, as the pre-clinic models using Conantokine-G as a NMDA receptor agonist to treat stroke have succeed, the pharmaceutic use has not been as expected (Balsara et al., 2012). Fort the treatment of seizures and epilepsy (Barton et al., 2004), Conantokine-G was proved with promising result in phase I, but failure in phase II of the clinical assays (obtained from a website called uniprot.org/uniprot/P07231,2014)
As there is no effective pharmacological treatment for regulating the processes involved in NMDA-dependent pathologies that has been found to date, therefore the search for new pharmacotherapy drugs directed against the different sites modulated by the NMDA receptor must continue.